Enzymes involved in squalene metabolism

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a squalene metabolic enzyme. The invention also relates to the construction of a chimeric gene encoding all or a portion of the squalene metabolic enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the squalene metabolic enzyme in a transformed host cell.

This application claims priority benefit of U.S. Provisional ApplicationNo. 60/105,405 filed Oct. 23, 1998, now pending.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingenzymes involved in squalene metabolism in plants and seeds.

BACKGROUND OF THE INVENTION

The terpenoids constitute the largest family of natural products withover 22,000 individual compounds of this class having been described.The terpenoids play diverse functional roles in plants as hormones,photosynthetic pigments, electron carriers, mediators of polysccharideassembly, and, structural components of membranes. Farnesylpyrophosphate is converted to squalene in the first dedicated steptowards sterol biosynthesis. Squalene is then converted tosqualene-2,3-epoxide which, in photosynthetic organisms, is convertedcycloarterenol.

Squalene monooxidase (EC 1.14.99.7), also referred to as squaleneepoxidase, is an oxidoreductase which acts on paired donors withincorporation of molecular oxygen. This enzyme is located at theendoplasmic reticulum, and catalyzes the conversion of squalene tosqualene 2,3-epoxide in the pathway to produce sterol. Squalenemonooxygenase may be the rate limiting step in sterol biosynthesis.Oxygen, NADPH, FAD, and a cytosolic protein are required for squalenemonooxygenase function. Squalene monooxygenase together with lanosterolsynthase was formerly known as squalene oxydocyclase.

Whereas vertebrates and fungi synthesize sterols from epoxysqualenethrough the intermediate lanosterol, plants cyclize epoxysqualene tocycloartenol as the initial sterol. This reaction is catalyzed bycycloartenol synthase (EC 5.4.99.8), also called2,3-epoxysqualene-cycloartenol cyclase.

Sequences encoding peptides with similarities to cycloartenol synthaseand squalene monooxygenase are found in the NCBI database having GeneralIdentifier Nos. 5566676, 5468642, 5706248, 5608422, 5055959, 2309779,2310417, 3107671, 702331, and 4874404.

SUMMARY OF THE INVENTION

The present invention relates to isolated polynucleotides comprising anucleotide sequence encoding a first polypeptide of at least 200 aminoacids that has at least 90% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of a corn cycloartenol synthase polypeptide of SEQ ID NO:2, arice cycloartenol synthase polypeptide of SEQ ID NO:4, a soybeancycloartenol synthase polypeptide of SEQ ID NO:6, and a wheatcycloartenol synthase polypeptide of SEQ ID NO:8. The present inventionalso relates to isolated polynucleotides comprising a nucleotidesequence encoding a first polypeptide of at least 200 amino acids thathas at least 90% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of a cornsqualene monooxygenase polypeptide of SEQ ID NO:10, a rice squalenemonooxygenase polypeptide of SEQ ID NO:12, a soybean squalenemonooxygenase polypeptide of SEQ ID NO:14, and a wheat squalenemonooxygenase polypeptide of SEQ ID NO:16. The present invention alsorelates to an isolated polynucleotide comprising the complement of thenucleotide sequences described above.

It is preferred that the isolated polynucleotides of the claimedinvention consist of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15 that codes for apolypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, and 16. The present invention also relates to an isolatedpolynucleotide comprising a nucleotide sequences of at least 40(preferably at least 30 or at least 15) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, and the complement of such nucleotidesequences.

The present invention relates to a chimeric gene comprising an isolatedpolynucleotide of the present invention operably linked to suitableregulatory sequences.

The present invention relates to an isolated host cell comprising achimeric gene of the present invention or an isolated polynucleotide ofthe present invention. The host cell may be eukaryotic, such as a yeastor a plant cell, or prokaryotic, such as a bacterial cell or virus. Ifthe host cell is a virus, it is preferably a baculovirus. A virus hostcell comprising an isolated polynucleotide of the present invention or achimeric gene of the present invention is most preferred.

The present invention relates to a process for producing an isolatedhost cell comprising a chimeric gene of the present invention or anisolated polynucleotide of the present invention, the process comprisingeither transforming or transfecting an isolated compatible host cellwith a chimeric gene or isolated polynucleotide of the presentinvention.

The present invention relates to a cycloartenol synthase polypeptide ofat least 200 amino acids comprising at least 90% homology based on theClustal method of alignment compared to a polypeptide selected from thegroup consisting of SEQ ID NOs:1, 4, 6, and 8. The present inventionrelates to a squalene monooxygenase polypeptide of at least 200 aminoacids comprising at least 90% homology based on the Clustal method ofalignment compared to a polypeptide selected from the group consistingof SEQ ID NOs:10, 12, 14, and 16.

The present invention relates to a method of selecting an isolatedpolynucleotide that affects the level of expression of a cycloartenolsynthase or a squalene monooxygenase polypeptide in a plant cell, themethod comprising the steps of:

constructing an isolated polynucleotide of the present invention or anisolated chimeric gene of the present invention;

introducing the isolated polynucleotide or the isolated chimeric geneinto a plant cell;

measuring the level a cycloartenol synthase or a squalene monooxygenasepolypeptide in the plant cell containing the isolated polynucleotide;and

comparing the level of a cycloartenol synthase or a squalenemonooxygenase polypeptide in the plant cell containing the isolatedpolynucleotide with the level of a cycloartenol synthase or a squalenemonooxygenase polypeptide in a plant cell that does not contain theisolated polynucleotide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a cycloartenol synthase or asqualene monooxygenase polypeptide gene, preferably a plant cycloartenolsynthase or a squalene monooxygenase polypeptide, comprising the stepsof: synthesizing an oligonucleotide primer comprising a nucleotidesequence of at least 40 (preferably at least 30 or at least 15)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and thecomplement of such nucleotide sequences; and amplifying a nucleic acidfragment (preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a portion of a cycloartenol synthase or a squalenemonooxygenase amino acid sequence.

The present invention also relates to a method of obtaining a nucleicacid fragment encoding all or a substantial portion of the amino acidsequence encoding a cycloartenol synthase or a squalene monooxygenasepolypeptide comprising the steps of: probing a cDNA or genomic librarywith an isolated polynucleotide of the present invention; identifying aDNA clone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

A further embodiment of the instant invention is a method for evaluatingat least one compound for its ability to inhibit the activity of acycloartenol synthase or a squalene monooxygenase, the method comprisingthe steps of: (a) transforming a host cell with a nucleic acid fragmentencoding; a cycloartenol synthase or a squalene monooxygenasepolypeptide preferably operably linked to suitable regulatory sequences;(b) growing the transformed host cell under conditions that are suitablefor expression of the nucleic acid fragment compound (such as theproduction of mRNA and/or polypeptide) wherein expression of the nucleicacid fragment preferably results in production of cycloartenol synthaseor squalene monooxygenase in the transformed host cell; (c) optionallypurifying the cycloartenol synthase or the squalene monooxygenaseexpressed by the transformed host cell; (d) treating the cycloartenolsynthase or the squalene monooxygenase with a compound to be tested; and(e) comparing the activity of the cycloartenol synthase or the squalenemonooxygenase that has been treated with a test compound to the activityof an untreated cycloartenol synthase or squalene monooxygenase, therebyselecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying Sequences Listing which form a part ofthis application.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Enzymes Involved in Squalene Metabolism SEQ ID NO: Protein CloneDesignation (Nucleotide) (Amino Acid) Corn cycloartenol synthasecep7.pk0019.f10 1 2 Rice cycloartenol synthase r1r2.pk0004.f6 3 4Soybean cycloartenol synthase sdp2c.pk008.g6 5 6 Wheat cycloartenolsynthase wr1.pk164.h10 7 8 Corn squalene monooxygenase Contig of: 9 10csi1n.pk0037.a8 p0045.ckdac10r p0083.cldb104r Rice squalenemonooxygenase Contig of: 11 12 res1c.pk006.o13 r10n.pk0031.d7 Soybeansqualene monooxygenase sdp3c.pk003.a5 13 14 Wheat squalene monooxygenasew1m1.pk0005.d6 15 16

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED: DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “polynucleotide” is a nucleotide sequence such as anucleic acid fragment. A polynucleotide any may be a polymer of RNA orDNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, or synthetic DNA. An isolated polynucleotide of thepresent invention may include at least 40 contiguous nucleotides,preferably at least 30 contiguous nucleotides, most preferably at least15 contiguous nucleotides, of the nucleic acid sequence of the SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, or 15.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides derived from the instantnucleic acid fragment can be constructed and introduced into a plant orplant cell. The level of the polypeptide encoded by the unmodifiednucleic acid fragment present in a plant or plant cell exposed to thesubstantially similar nucleic fragment can then be compared to the levelof the polypeptide in a plant or plant cell that is not exposed to thesubstantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 40 (preferably at least 30, mostpreferably at least 15) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15, and the complement of such nucleotide sequences may be usedin methods of selecting an isolated polynucleotide that affects theexpression of a polypeptide (such as cycloartenol synthase or squalenemonooxygenase) in a host cell. A method of selecting an isolatedpolynucleotide that affects the level of expression of a polypeptide ina host cell (eukaryotic, such as plant, or prokarotic such as yeastbacterial or virus) may comprise the steps of: constructing an isolatedpolynucleotide of the present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or theisolated chimeric gene into a host cell; measuring the level thepolypeptide in the host cell containing the isolated polynucleotide; andcomparing the level of the polypeptide in the host cell containing theisolated polynucleotide with the level of the polypeptide in a host cellthat does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are 85% identical to theamino acid sequences reported herein. Preferably 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Suitable nucleic acid fragmentsnot only have the above identities but typically encode a polypeptidehaving at least 50 amino acids, preferably 100 amino acids, morepreferably 150 amino acids, still more preferably 200 amino acids, andmost preferably 250 amino acids. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used: as amplification primers in PCRin order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to j as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene an,d the codingsequence. The translation leader sequence is present in the fullyprocessed mRNA upstream of the translation start sequence. Thetranslation leader sequence may affect processing of the primarytranscript to mRNA, mRNA stability or translation efficiency. Examplesof translation leader sequences have been described (Turner and Foster(1995) Mol. Biotechnol. 3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA, precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA,) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several enzymesinvolved in squalene metabolism have bee h isolated and identified bycomparison of random plant CDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto,: methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other cycloartenol synthases or squalenemonooxygenases, either as cDNAs or genomic DNAs, could be isolateddirectly by using all or a portion of the instant nucleic acid fragmentsas DNA hybridization probes to screen libraries from any desired plantemploying methodology well known to those skilled in the art. Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis).Moreover, the entire sequences can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primer DNAlabeling, nick translation, or end-labeling techniques, or RNA probesusing available in vitro transcription systems. In addition, specificprimers can be designed and used to amplify a part or all of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast 40 (preferably at least 30, most preferably at least 15)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and thecomplement of such nucleotide sequences may be used in such methods toobtain a nucleic acid fragment encoding a substantial portion of anamino acid sequence of a polypeptide. The present invention relates to amethod of obtaining a nucleic acid fragment encoding a substantialportion of a polypeptide of a gene (such as cycloartenol synthase orsqualene monooxygenase) preferably a substantial portion of a plantpolypeptide, comprising the steps of: synthesizing an oligonucleotideprimer comprising a nucleotide sequence of at least 40 (preferably atleast 30, most preferably at least 15) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, and the complement of such nucleotidesequences; and amplifying a nucleic acid fragment (preferably a cDNAinserted in a cloning vector) using the oligonucleotide primer. Theamplified nucleic acid fragment preferably will encode a portion of apolypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen CDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.336:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the relative sterol composition in those cells. Thesechanges in the plant seed may be useful to improve the seed nutritionalvalue, and in the plant leaf may aid in insect tolerance. Squalenemonooxygenase catalyzes one of the rate limiting steps in squalenebiosynthesis and, thus, is a good herbicide target. Catalyzing an earlystep in sterol synthesis, squalene synthase catalyzes a required step inthe synthesis of saponins in soybean seeds. Elimination of saponinsmight lead to improved flavor.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate theirsecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53,), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds, and is not an inherentpart of the invention. For example, one can screen by looking forchanges in gene expression by using antibodies specific for the proteinencoded by the gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded enzyme involved in squalene metabolism. An example of avector for high level expression of the instant polypeptides in abacterial host is provided (Example 7).

Additionally, the instant polypeptides can be used as targets tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze various steps in squalenebiosynthesis. Accordingly, inhibition of the activity of one or more ofthe enzymes described herein could lead to inhibition of plant growth.Thus, the instant polypeptides could be appropriate for new herbicidediscovery and design.

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses, using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153, Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean, andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean, and Wheat LibraryTissue Clone cep7 Corn 7 Day Old Epicotyl; Grown in Lightcep7.pk0019.f10 csi1n Corn Silk* csi1n.pk0037.a8 p0045 Hi-II SuspensionCulture p0045.ckdac10r p0083 Corn Whole Kernels 7 Days After Pollinationp0083.cldb104r res1c Rice Etiolated Seedling res1c.pk006.o13 r10n Rice15 Day Old Leaf* r10n.pk0031.d7 r1r2 Rice Leaf 15 Days AfterGermination, 2 Hours After r1r2.pk0004.f6 Infection of Strain Magaporthegrisea 4360-R-62 (AVR2-YAMO); Resistant sdp2c Soybean Developing Pods(6-7 mm) sdp2c.pk008.g6 sdp3c Soybean Developing Pods (8-9 mm)sdp3c.pk003.a5 w1m1 Wheat Seedlings 1 Hour After Inoculation WithErysiphe w1m1.pk0005.d6 graminis f. sp tritici wr1 Wheat Root From 7 DayOld Seedling wr1.pk164.h10 *These libraries were normalized essentiallyas described in U.S. Pat. No. 5,482,845, incorporated herein byreference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (19911) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding enzymes involved in squalene metabolism wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Cycloartenol Synthase

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs tocycloartenol synthase from Glycyrrhiza glabra (NCBI General IdentifierNo. 4589852). Shown in Table 3 are the BLAST results for the sequencesof the entire cDNA inserts comprising the indicated cDNA clones (“FIS”),or sequences en coding the entire protein derived from an FIS (“CGS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toCycloartenol Synthase BLAST pLog Score Clone Status 4589852cep7.pk0019.f10 CGS >254.00 r1r2.pk0004.f6 FIS >254.00 sdp2c.pk008.g6CGS >254.00 wr1.pk164.h10 FIS 160.0

The data in Table 4 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 4, 6 and 8 and theGlycyrrhiza glabra sequence (NCBI General Identifier No. 4589852).

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toCycloartenol Synthase Percent Identity to SEQ ID NO. 4589852 2 78.2 477.8 6 90.4 8 75.1

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a corn, a rice, a soybean and a wheatcycloartenol synthase. These sequences represent the first corn, rice,soybean, and wheat sequences encoding cycloartenol synthase.

Example 4 Characterization of cDNA Clones Encoding SqualeneMonooxygenase

The BLASTX search using the EST sequences from clones listed in Table 5revealed similarity of the polypeptides encoded by the cDNAs to squalenemonooxygenase from Panax ginseng (NCBI General Identifier No. 2804278).Shown in Table 5 are the BLAST results for the sequences of the entirecDNA inserts comprising the indicated cDNA clones (“FIS”), contigsassembled from an FIS and one or more ESTs (“Contig*”), or sequencesencoding the entire protein derived from an FIS (“CGS”):

TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous toSqualene Monooxygenase BLAST pLog Score Clone Status 2804278 Contig of:Contig* >254.00 csi1n.pk0037.a8 p0045.ckdac10r p0083.cldb104r Contig of:Contig* 161.00 resc.pk006.o13 r10n.pk0031.d7 sdp3c.pk003.a5 CGS >254.00w1m1.pk0005.d6 FIS >254.00

The data in Table 6 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:10, 12, 14 and 16 andthe Panax ginseng sequence (NCBI General Identifier No. 2804278).

TABLE 6 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toSqualene Monooxygenase Percent Identity to SEQ ID NO. 2804278 10 81.9 1280.6 14 75.7 16 83.7

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of,a corn, a rice,. a soybean, and a wheatsqualene monooxygenase. These sequences represent the first corn, rice,soybean, and wheat sequences encoding squalene monooxygenase.

Example 5 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the CDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML 103. Plasmid pML 103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL 1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LHI 32. The embryos are isolated 10 to 11 days after pollinationwhen they are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment: method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotideshupstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC 18 vector carrying theseed expression cassette.

Soybean embroys may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/F7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 8 Evaluating Compounds for their Ability to Inhibit the Activityof Enzymes Involved in Squalene Metabolism

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria as described in Example 7, orexpression in eukaryotic cell culture, in plant a, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant polypeptides may be expressed either as mature forms of theproteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

Purification of the instants polypeptides, if desired, may utilize anynumber of separation technologies familiar to those skilled in the artof protein purification. Examples of such methods include, but are notlimited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose4B. In an alternate embodiment, athioredoxin fusion protein may be eluted using dithiothreitol; however,elution may be accomplished using other reagents which interact todisplace the thioredoxin from the resin. These reagents includeP-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

Crude, partially purified or purified enzyme, either alone or as afusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. For example, assays for cycloartenol synthase are presented byMorita et al. (1997) Biol. Pharm. Bull. 20:770-775. Assays for squalenemonooxygenase are presented by Grieveson et al. (1997) Anal. Biochem.252:19-23.

16 1 2558 DNA Zea mays 1 gcacgagcaa gcacagcgcc gacctcctca tgcgcatccagttcgccaaa gaaaactcga 60 ttgagcttca ccttccaggc atcaagctcg gtgagcatgaagatgtgacc gaggaagctg 120 tgttgactac attgaaaagg gcaatcagcc gtttctctactctccaggca catgatggac 180 actggcctgg ggattatggt ggtcctatgt tccttatgccaggcttgatc ataacattgt 240 atgtgactgg agcactaaac actgtcttgt cattggaacaccagaaggag atccgccggt 300 atctttataa tcaccagaat gaagatggcg gctggggcttgcacattgag ggtccaagca 360 ccatgttcgg ctcagcactg acctatgtta ttttgagattgcttggagag ggaccagata 420 gtggagatgg agccatggag aaaggtcgaa actggatattagaccatggt ggagcaacat 480 atataacatc gtgggggaag ttttggcttt cggtactaggtgtatttgaa tggtctggta 540 acaacccggt gccaccagaa gtatggctac tgccatatctcctcccattt cacccaggga 600 ggatgtggtg tcattgtcga atggtgtatt tgccaatgtgctacatttat gggaagaggt 660 ttgttggccg aatcacacca cttctgttgg aattaaggaaggaacttttc aaagaccctt 720 acagcaagat tgattgggac aaggcccgca acctatgtgccaaggaagat ctgtactacc 780 cacacccatt cgttcaagat gtgttgtggg ccactctccataaattcgtt gaaccagtta 840 tgatgcattg gcctggcagc aaattgaggg agaaagctctggaaacagtc atgcaacatg 900 ttcattatga agatgagaac actcgttata tttgcattggtcctgtaaac aaggtattga 960 atatgcttgc ttgctggatt gaagatccaa actcggaggccttcaaactt catatcccac 1020 gagtctatga ttacttgtgg cttgctgaag atggcatgaagatgcagggt tataatggca 1080 gccaactttg ggatacagct ttcacagttc aagccattgtggctaccaac cttattgaag 1140 agtttggtcc tacccttaaa ctagcacaca actatatcaagaattcacag gttcttgatg 1200 actgccctgg tgatctgaat gactggtacc gccacacatctaaaggtgca tggccattct 1260 caactgctga tcatggttgg cctatatctg attgcactgctgaaggacta aaggcttcat 1320 tattgttatc aaggatctct cccaaaattg ttggtgaaccgatggaagct aatagatttt 1380 atgatgctgt cagttgtctg atgtcttata tgaatgataatggcggtttc gcgacatatg 1440 aactcacaag atcttatccc tggttggagc tgatcaatcccgctgagacc tttggggata 1500 ttgtgattga ttacccgtat gttgaatgta catcagcagcaattcaggcc ctgacatcat 1560 tcaaaaaact ataccctggg caccgcagga aagaggtggataactgtatc agcaaagctt 1620 ccaatttcat cgagagtatt cagaaaagcg atggttcatggtatggctct tgggccgtct 1680 gtttcacata cggcacttgg tttggtgtga agggactaattgctgctggt agaacatttg 1740 agaacagtcc tgcaattaga aaggcatgcg actttctgttgtcaaaagaa cttccttccg 1800 gtggttgggg agaaagctat ttgtcatctc aagaccaggtttacaccaat ctcaaaggca 1860 accggcctca tgcggtgaac actagttggg ccatgctggcgctgattgat gcgggccagg 1920 ccgagagaga tccaacgcct ctacaccgag cagcaaaggttttgatcaac ttacaatcag 1980 aggacggaga atttcctcag caagagatca taggagtgttcaacaagaac tgcatgataa 2040 gctactccca gtacaggaac atcttcccga tttgggctctgggtgagtac cggtgtcgag 2100 tcttgggggc tggcaagcct tggcggtgaa cgggaggtgtgtgtgtgtgt gtgtcatgga 2160 tcagcttttg tgagtagcca tgtggaagtt ggaataatgtagctacgtta cgttcagggg 2220 gttgcgttac tagtggtcct agtaataata gtgatggtgatagtaatgta ctcctcatta 2280 ttacaatctc aaagcggttc atgccattgc catgcacatctcagatccga gtcacgcact 2340 tgagagagtt caaggattgc aagtatagtt gggagaatcaaatccaatcg gcttattgtc 2400 tgctcatctc aggtgtcagg tctttcagcc acacacacatacacataccc tgacctagag 2460 atttttgcca ttatgaaaca ttacatgttc ggcttcgttgaaatgaagat gagaagggat 2520 tcgacgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa2558 2 701 PRT Zea mays 2 Thr Ser Lys His Ser Ala Asp Leu Leu Met ArgIle Gln Phe Ala Lys 1 5 10 15 Glu Asn Ser Ile Glu Leu His Leu Pro GlyIle Lys Leu Gly Glu His 20 25 30 Glu Asp Val Thr Glu Glu Ala Val Leu ThrThr Leu Lys Arg Ala Ile 35 40 45 Ser Arg Phe Ser Thr Leu Gln Ala His AspGly His Trp Pro Gly Asp 50 55 60 Tyr Gly Gly Pro Met Phe Leu Met Pro GlyLeu Ile Ile Thr Leu Tyr 65 70 75 80 Val Thr Gly Ala Leu Asn Thr Val LeuSer Leu Glu His Gln Lys Glu 85 90 95 Ile Arg Arg Tyr Leu Tyr Asn His GlnAsn Glu Asp Gly Gly Trp Gly 100 105 110 Leu His Ile Glu Gly Pro Ser ThrMet Phe Gly Ser Ala Leu Thr Tyr 115 120 125 Val Ile Leu Arg Leu Leu GlyGlu Gly Pro Asp Ser Gly Asp Gly Ala 130 135 140 Met Glu Lys Gly Arg AsnTrp Ile Leu Asp His Gly Gly Ala Thr Tyr 145 150 155 160 Ile Thr Ser TrpGly Lys Phe Trp Leu Ser Val Leu Gly Val Phe Glu 165 170 175 Trp Ser GlyAsn Asn Pro Val Pro Pro Glu Val Trp Leu Leu Pro Tyr 180 185 190 Leu LeuPro Phe His Pro Gly Arg Met Trp Cys His Cys Arg Met Val 195 200 205 TyrLeu Pro Met Cys Tyr Ile Tyr Gly Lys Arg Phe Val Gly Arg Ile 210 215 220Thr Pro Leu Leu Leu Glu Leu Arg Lys Glu Leu Phe Lys Asp Pro Tyr 225 230235 240 Ser Lys Ile Asp Trp Asp Lys Ala Arg Asn Leu Cys Ala Lys Glu Asp245 250 255 Leu Tyr Tyr Pro His Pro Phe Val Gln Asp Val Leu Trp Ala ThrLeu 260 265 270 His Lys Phe Val Glu Pro Val Met Met His Trp Pro Gly SerLys Leu 275 280 285 Arg Glu Lys Ala Leu Glu Thr Val Met Gln His Val HisTyr Glu Asp 290 295 300 Glu Asn Thr Arg Tyr Ile Cys Ile Gly Pro Val AsnLys Val Leu Asn 305 310 315 320 Met Leu Ala Cys Trp Ile Glu Asp Pro AsnSer Glu Ala Phe Lys Leu 325 330 335 His Ile Pro Arg Val Tyr Asp Tyr LeuTrp Leu Ala Glu Asp Gly Met 340 345 350 Lys Met Gln Gly Tyr Asn Gly SerGln Leu Trp Asp Thr Ala Phe Thr 355 360 365 Val Gln Ala Ile Val Ala ThrAsn Leu Ile Glu Glu Phe Gly Pro Thr 370 375 380 Leu Lys Leu Ala His AsnTyr Ile Lys Asn Ser Gln Val Leu Asp Asp 385 390 395 400 Cys Pro Gly AspLeu Asn Asp Trp Tyr Arg His Thr Ser Lys Gly Ala 405 410 415 Trp Pro PheSer Thr Ala Asp His Gly Trp Pro Ile Ser Asp Cys Thr 420 425 430 Ala GluGly Leu Lys Ala Ser Leu Leu Leu Ser Arg Ile Ser Pro Lys 435 440 445 IleVal Gly Glu Pro Met Glu Ala Asn Arg Phe Tyr Asp Ala Val Ser 450 455 460Cys Leu Met Ser Tyr Met Asn Asp Asn Gly Gly Phe Ala Thr Tyr Glu 465 470475 480 Leu Thr Arg Ser Tyr Pro Trp Leu Glu Leu Ile Asn Pro Ala Glu Thr485 490 495 Phe Gly Asp Ile Val Ile Asp Tyr Pro Tyr Val Glu Cys Thr SerAla 500 505 510 Ala Ile Gln Ala Leu Thr Ser Phe Lys Lys Leu Tyr Pro GlyHis Arg 515 520 525 Arg Lys Glu Val Asp Asn Cys Ile Ser Lys Ala Ser AsnPhe Ile Glu 530 535 540 Ser Ile Gln Lys Ser Asp Gly Ser Trp Tyr Gly SerTrp Ala Val Cys 545 550 555 560 Phe Thr Tyr Gly Thr Trp Phe Gly Val LysGly Leu Ile Ala Ala Gly 565 570 575 Arg Thr Phe Glu Asn Ser Pro Ala IleArg Lys Ala Cys Asp Phe Leu 580 585 590 Leu Ser Lys Glu Leu Pro Ser GlyGly Trp Gly Glu Ser Tyr Leu Ser 595 600 605 Ser Gln Asp Gln Val Tyr ThrAsn Leu Lys Gly Asn Arg Pro His Ala 610 615 620 Val Asn Thr Ser Trp AlaMet Leu Ala Leu Ile Asp Ala Gly Gln Ala 625 630 635 640 Glu Arg Asp ProThr Pro Leu His Arg Ala Ala Lys Val Leu Ile Asn 645 650 655 Leu Gln SerGlu Asp Gly Glu Phe Pro Gln Gln Glu Ile Ile Gly Val 660 665 670 Phe AsnLys Asn Cys Met Ile Ser Tyr Ser Gln Tyr Arg Asn Ile Phe 675 680 685 ProIle Trp Ala Leu Gly Glu Tyr Arg Cys Arg Val Leu 690 695 700 3 1882 DNAOryza sativa 3 gcacgagatc actgctttcg gtgcttggtg tatttgactg gtctggcaacaacccagtgc 60 caccagaaat atggttgttg ccatatttcc tgccgattca tccagggcgaatgtggtgtc 120 attgccggat ggtttatttg cctatgtgtt acatttatgg aaagaggtttgtgggcccag 180 ttacaccaat tatattggaa ttaagaaagg aactctacga agtaccctacaatgaagttg 240 attgggacaa ggctcgcaat ctatgtgcta aggaagatct gtactatccacatccattcg 300 tgcaggatgt attatgggcc actctccaca aatttgttga accagctatgttgcgttggc 360 ctgggaacaa attgagggag aaagctttgg acactgtcat gcagcatattcattatgaag 420 atgagaacac ccgatatatt tgcattggtc cagtaaacaa ggtattaaatatgcttgctt 480 gctggattga agatccaaac tcagaggcat tcaaactcca cattccaagagtccacgatt 540 acctatggat tgcagaagat ggcatgaaaa tgcagggtta taatggaagccagctgtggg 600 acacagcttt cacagttcaa gctatagtgg ctactggcct cattgaagaatttggtccta 660 ctcttaaact agcacatggc tacataaaga aaacgcaggt tatcgatgactgccctggag 720 atcttagtca gtggtaccgc cacatatcta aaggtgcatg gcccttttctactgctgatc 780 atggttggcc tatatcagat tgcactgcag aaggacttaa ggcggcattattgctatcga 840 agatttctcc agatattgtt ggcgaagcag tggaagttaa tagactgtatgattctgtca 900 attgtttgat gtcatacatg aatgataatg gtggatttgc aacatatgaactcacaaggt 960 cttatgcctg gctggagctt atcaatcctg ctgagacctt tggggacattgtgattgatt 1020 atccttatgt ggaatgcact tcagcagcaa ttcaggccct gacagcatttaaaaagctct 1080 accctggaca ccgcaagagt gaaatagaca actgtataag caaagctgctagctttattg 1140 agggtattca aaaaagcgat ggttcatggt atggttcttg ggctgtttgttttacctatg 1200 gcacatggtt tggtgtaaag ggattagttg ctgctggtag gacattcaaaaacagtcctg 1260 caatcagaaa ggcatgtgac tttttgttgt caaaagagct tccttctggaggctggggag 1320 aaagctattt gtcatcccaa gatcaggttt ataccaatct cgaagggaagcgacctcatg 1380 cggtgaacac tggttgggcc atgctagccc taatcgatgc agggcaggctgagagagatc 1440 caattccttt gcatcgagca gcgaaggttt tgatcaactt acaatcggaagatggtgaat 1500 ttccccagca agagatcatt ggagtcttca acaaaaactg catgatcagctactccgagt 1560 atagaaacat cttccctatt tgggcccttg gggagtaccg tcgccgcgtcttggccgcag 1620 acaagtagtt cagcacgagc agagcagcag caccaacaat gtgcatgtatttatacgtga 1680 aataatgtag ctatgtttca gttgtaataa tgtggctata tgtattctcccgttagtgat 1740 gccacgcgag cgtagtcaaa tagaaacgca ttttgacaca agttcgagatgaatggattc 1800 ctgaatcgaa tgtttgtgtt caaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 1860 aaaaaaaaaa aaaaaaaaaa cc 1882 4 541 PRT Oryza sativa 4Thr Arg Ser Leu Leu Ser Val Leu Gly Val Phe Asp Trp Ser Gly Asn 1 5 1015 Asn Pro Val Pro Pro Glu Ile Trp Leu Leu Pro Tyr Phe Leu Pro Ile 20 2530 His Pro Gly Arg Met Trp Cys His Cys Arg Met Val Tyr Leu Pro Met 35 4045 Cys Tyr Ile Tyr Gly Lys Arg Phe Val Gly Pro Val Thr Pro Ile Ile 50 5560 Leu Glu Leu Arg Lys Glu Leu Tyr Glu Val Pro Tyr Asn Glu Val Asp 65 7075 80 Trp Asp Lys Ala Arg Asn Leu Cys Ala Lys Glu Asp Leu Tyr Tyr Pro 8590 95 His Pro Phe Val Gln Asp Val Leu Trp Ala Thr Leu His Lys Phe Val100 105 110 Glu Pro Ala Met Leu Arg Trp Pro Gly Asn Lys Leu Arg Glu LysAla 115 120 125 Leu Asp Thr Val Met Gln His Ile His Tyr Glu Asp Glu AsnThr Arg 130 135 140 Tyr Ile Cys Ile Gly Pro Val Asn Lys Val Leu Asn MetLeu Ala Cys 145 150 155 160 Trp Ile Glu Asp Pro Asn Ser Glu Ala Phe LysLeu His Ile Pro Arg 165 170 175 Val His Asp Tyr Leu Trp Ile Ala Glu AspGly Met Lys Met Gln Gly 180 185 190 Tyr Asn Gly Ser Gln Leu Trp Asp ThrAla Phe Thr Val Gln Ala Ile 195 200 205 Val Ala Thr Gly Leu Ile Glu GluPhe Gly Pro Thr Leu Lys Leu Ala 210 215 220 His Gly Tyr Ile Lys Lys ThrGln Val Ile Asp Asp Cys Pro Gly Asp 225 230 235 240 Leu Ser Gln Trp TyrArg His Ile Ser Lys Gly Ala Trp Pro Phe Ser 245 250 255 Thr Ala Asp HisGly Trp Pro Ile Ser Asp Cys Thr Ala Glu Gly Leu 260 265 270 Lys Ala AlaLeu Leu Leu Ser Lys Ile Ser Pro Asp Ile Val Gly Glu 275 280 285 Ala ValGlu Val Asn Arg Leu Tyr Asp Ser Val Asn Cys Leu Met Ser 290 295 300 TyrMet Asn Asp Asn Gly Gly Phe Ala Thr Tyr Glu Leu Thr Arg Ser 305 310 315320 Tyr Ala Trp Leu Glu Leu Ile Asn Pro Ala Glu Thr Phe Gly Asp Ile 325330 335 Val Ile Asp Tyr Pro Tyr Val Glu Cys Thr Ser Ala Ala Ile Gln Ala340 345 350 Leu Thr Ala Phe Lys Lys Leu Tyr Pro Gly His Arg Lys Ser GluIle 355 360 365 Asp Asn Cys Ile Ser Lys Ala Ala Ser Phe Ile Glu Gly IleGln Lys 370 375 380 Ser Asp Gly Ser Trp Tyr Gly Ser Trp Ala Val Cys PheThr Tyr Gly 385 390 395 400 Thr Trp Phe Gly Val Lys Gly Leu Val Ala AlaGly Arg Thr Phe Lys 405 410 415 Asn Ser Pro Ala Ile Arg Lys Ala Cys AspPhe Leu Leu Ser Lys Glu 420 425 430 Leu Pro Ser Gly Gly Trp Gly Glu SerTyr Leu Ser Ser Gln Asp Gln 435 440 445 Val Tyr Thr Asn Leu Glu Gly LysArg Pro His Ala Val Asn Thr Gly 450 455 460 Trp Ala Met Leu Ala Leu IleAsp Ala Gly Gln Ala Glu Arg Asp Pro 465 470 475 480 Ile Pro Leu His ArgAla Ala Lys Val Leu Ile Asn Leu Gln Ser Glu 485 490 495 Asp Gly Glu PhePro Gln Gln Glu Ile Ile Gly Val Phe Asn Lys Asn 500 505 510 Cys Met IleSer Tyr Ser Glu Tyr Arg Asn Ile Phe Pro Ile Trp Ala 515 520 525 Leu GlyGlu Tyr Arg Arg Arg Val Leu Ala Ala Asp Lys 530 535 540 5 2560 DNAGlycine max 5 ttggcctctt gccagcaaaa cagaatgtgg aagctcaagt tcgccgaaggagggaatcca 60 tggcttcgga cattgaacaa tcacgttgga agacaggtgt gggagttcgatcctaagctt 120 ggatcgccgc aagatctcct cgagattgag aaagctcgcc agaattttcacgataaccgc 180 tttacccaca aacacagcgc tgatctactt atgcggatgc agttcgcaagagagaaccca 240 acacgtgaag tcttgcccaa agtcggagtt aaggatattg aggatgtgacccaagagatt 300 gtgacaaaaa cattaagaag ggccgtaagt ttccattcaa ctctccagtgccatgacgga 360 cactggccgg gagattatgg aggtcccatg tttctgatgc ctggcttggtaattactctg 420 tctatcactg gggcgttgaa tacagtctta actgaagaac atagaaaggaaatatgccgt 480 tacctctata atcatcaaaa caaggatggt gggtggggtt tgcatattgaaggtccaagc 540 accatgtttg gctctgtctt gagttatatt actctgagat tgctaggtgaggggcctaat 600 gatggacaag gggaaatgga gaaggcacgt gactggattc tagggcatggtggtgctact 660 tatataacgt catgggggaa gatgtggctt tcagtacttg gagtgtatgaatggtctgga 720 aataatcccc tgccccctga gatatggctc cttccataca tgcttccatttcatccagga 780 aggatgtggt gtcactgccg gatggtctat ttgccgatgt cctacttatatggcaagagg 840 tttgttggtc caatctcacc aacagtatta tctttgagaa aagagctttatacagtacca 900 taccatgata tagattggga tcaggctcgc aatttgtgtg caaaggaagatttgtactat 960 cctcacccac ttgtacagga tattctttgg gcatctctac acaagttccttgagcctatt 1020 ctgatgcatt ggcctggaaa aagattgagg gaaaaggcta ttatttctgcattggagcat 1080 atacattacg aagatgagaa tactcgatat atttgcatag gtcctgtaaataaggtgtta 1140 aatatgcttt gctgttgggt ggaagatcca aattctgagg ccttcaagttgcatcttccc 1200 aggatttatg attatctatg gattgcagaa gatggcatga aaatgcagggctacaatgga 1260 agtcaactat gggacactgc ttttgctgtc caagcaatta ttgcatctaacctcattgaa 1320 gaatttggtc caactataag aaaagctcat acctatatta agaattcacaggttttagaa 1380 gattgtccag gtgatcttaa taaatggtac cgtcacattt caaaaggtgcttggcctttt 1440 tcaactggag atcatggatg gccaatttct gactgcacag ctgaaggactgaaagctgtt 1500 ctattactat ccaaaattgc accagaaata gttggtgagc caatagacgtgaagcgatta 1560 tatgattctg taaatgtcat tctctcacta cagaatgaag atggtggttttgcaacatat 1620 gagcttaaac gatcttataa ttggttggag ataatcaatc ctgctgaaacttttggtgac 1680 atcgttattg attatcctta tgtggaatgt acatcagcag cgattcaagctttggcatca 1740 tttaggaaat tatatcctgg gcatcgccga gaagaaatac aacattgtatcgataaagcc 1800 actaccttca ttgaaaaaat acaagcttca gatggatcat ggtatggttcttggggagtt 1860 tgcttcactt acggtgcttg gtttggggta aaaggtctga ttgctgctggaaggagtttc 1920 agtaattgct caagcatccg taaagcttgt gaatttctgc tgtccaagcagcttccttct 1980 ggtggctggg gagagagtta tctgtcctgt caaaacaagg tgtattcaaatctggaaggc 2040 aacaggtctc atgtggtcaa cactgggtgg gctatgttgg ctctcattgatgctggacag 2100 gctaagagag attcgcaacc actgcaccgg gcagctgcat acttgataaattcccaattg 2160 gaggacggtg actttccgca gcaggaaata atgggagtct tcaacaagaattgcatgatc 2220 acatacgccg catacagaaa catattcccc atttgggcgt tgggagaataccaatcccaa 2280 gtattgcaat ctcgttaatc gagccttagt tagggtgtca tcctaattatttcgacctgt 2340 ggacagtaaa agaaataata ataataacct atttttcttt tatttccatggctctcttaa 2400 aatgtttgtg actaatgagt ttagtagtca gctaaaaaaa aaagcaaacacgtggagaat 2460 gcctgtaagt tttttctatt actcatagac gcctctcttg ctttccctgcagcagaggaa 2520 ttaaatatat acataaatag agatataaaa aaaaaaaaaa 2560 6 757PRT Glycine max 6 Met Trp Lys Leu Lys Phe Ala Glu Gly Gly Asn Pro TrpLeu Arg Thr 1 5 10 15 Leu Asn Asn His Val Gly Arg Gln Val Trp Glu PheAsp Pro Lys Leu 20 25 30 Gly Ser Pro Gln Asp Leu Leu Glu Ile Glu Lys AlaArg Gln Asn Phe 35 40 45 His Asp Asn Arg Phe Thr His Lys His Ser Ala AspLeu Leu Met Arg 50 55 60 Met Gln Phe Ala Arg Glu Asn Pro Thr Arg Glu ValLeu Pro Lys Val 65 70 75 80 Gly Val Lys Asp Ile Glu Asp Val Thr Gln GluIle Val Thr Lys Thr 85 90 95 Leu Arg Arg Ala Val Ser Phe His Ser Thr LeuGln Cys His Asp Gly 100 105 110 His Trp Pro Gly Asp Tyr Gly Gly Pro MetPhe Leu Met Pro Gly Leu 115 120 125 Val Ile Thr Leu Ser Ile Thr Gly AlaLeu Asn Thr Val Leu Thr Glu 130 135 140 Glu His Arg Lys Glu Ile Cys ArgTyr Leu Tyr Asn His Gln Asn Lys 145 150 155 160 Asp Gly Gly Trp Gly LeuHis Ile Glu Gly Pro Ser Thr Met Phe Gly 165 170 175 Ser Val Leu Ser TyrIle Thr Leu Arg Leu Leu Gly Glu Gly Pro Asn 180 185 190 Asp Gly Gln GlyGlu Met Glu Lys Ala Arg Asp Trp Ile Leu Gly His 195 200 205 Gly Gly AlaThr Tyr Ile Thr Ser Trp Gly Lys Met Trp Leu Ser Val 210 215 220 Leu GlyVal Tyr Glu Trp Ser Gly Asn Asn Pro Leu Pro Pro Glu Ile 225 230 235 240Trp Leu Leu Pro Tyr Met Leu Pro Phe His Pro Gly Arg Met Trp Cys 245 250255 His Cys Arg Met Val Tyr Leu Pro Met Ser Tyr Leu Tyr Gly Lys Arg 260265 270 Phe Val Gly Pro Ile Ser Pro Thr Val Leu Ser Leu Arg Lys Glu Leu275 280 285 Tyr Thr Val Pro Tyr His Asp Ile Asp Trp Asp Gln Ala Arg AsnLeu 290 295 300 Cys Ala Lys Glu Asp Leu Tyr Tyr Pro His Pro Leu Val GlnAsp Ile 305 310 315 320 Leu Trp Ala Ser Leu His Lys Phe Leu Glu Pro IleLeu Met His Trp 325 330 335 Pro Gly Lys Arg Leu Arg Glu Lys Ala Ile IleSer Ala Leu Glu His 340 345 350 Ile His Tyr Glu Asp Glu Asn Thr Arg TyrIle Cys Ile Gly Pro Val 355 360 365 Asn Lys Val Leu Asn Met Leu Cys CysTrp Val Glu Asp Pro Asn Ser 370 375 380 Glu Ala Phe Lys Leu His Leu ProArg Ile Tyr Asp Tyr Leu Trp Ile 385 390 395 400 Ala Glu Asp Gly Met LysMet Gln Gly Tyr Asn Gly Ser Gln Leu Trp 405 410 415 Asp Thr Ala Phe AlaVal Gln Ala Ile Ile Ala Ser Asn Leu Ile Glu 420 425 430 Glu Phe Gly ProThr Ile Arg Lys Ala His Thr Tyr Ile Lys Asn Ser 435 440 445 Gln Val LeuGlu Asp Cys Pro Gly Asp Leu Asn Lys Trp Tyr Arg His 450 455 460 Ile SerLys Gly Ala Trp Pro Phe Ser Thr Gly Asp His Gly Trp Pro 465 470 475 480Ile Ser Asp Cys Thr Ala Glu Gly Leu Lys Ala Val Leu Leu Leu Ser 485 490495 Lys Ile Ala Pro Glu Ile Val Gly Glu Pro Ile Asp Val Lys Arg Leu 500505 510 Tyr Asp Ser Val Asn Val Ile Leu Ser Leu Gln Asn Glu Asp Gly Gly515 520 525 Phe Ala Thr Tyr Glu Leu Lys Arg Ser Tyr Asn Trp Leu Glu IleIle 530 535 540 Asn Pro Ala Glu Thr Phe Gly Asp Ile Val Ile Asp Tyr ProTyr Val 545 550 555 560 Glu Cys Thr Ser Ala Ala Ile Gln Ala Leu Ala SerPhe Arg Lys Leu 565 570 575 Tyr Pro Gly His Arg Arg Glu Glu Ile Gln HisCys Ile Asp Lys Ala 580 585 590 Thr Thr Phe Ile Glu Lys Ile Gln Ala SerAsp Gly Ser Trp Tyr Gly 595 600 605 Ser Trp Gly Val Cys Phe Thr Tyr GlyAla Trp Phe Gly Val Lys Gly 610 615 620 Leu Ile Ala Ala Gly Arg Ser PheSer Asn Cys Ser Ser Ile Arg Lys 625 630 635 640 Ala Cys Glu Phe Leu LeuSer Lys Gln Leu Pro Ser Gly Gly Trp Gly 645 650 655 Glu Ser Tyr Leu SerCys Gln Asn Lys Val Tyr Ser Asn Leu Glu Gly 660 665 670 Asn Arg Ser HisVal Val Asn Thr Gly Trp Ala Met Leu Ala Leu Ile 675 680 685 Asp Ala GlyGln Ala Lys Arg Asp Ser Gln Pro Leu His Arg Ala Ala 690 695 700 Ala TyrLeu Ile Asn Ser Gln Leu Glu Asp Gly Asp Phe Pro Gln Gln 705 710 715 720Glu Ile Met Gly Val Phe Asn Lys Asn Cys Met Ile Thr Tyr Ala Ala 725 730735 Tyr Arg Asn Ile Phe Pro Ile Trp Ala Leu Gly Glu Tyr Gln Ser Gln 740745 750 Val Leu Gln Ser Arg 755 7 1300 DNA Triticum aestivum 7gcacgaggac acagcttttg cagttcaagc tattgcggcc actgacctca ttgaagagtt 60tgctcccact cttaagctgg cacatgattt tattaagaac tctcaggttg ttgatgactg 120ccctggagat ctgagttact ggtaccgtca catatctaaa ggtgcatggc ccttttctac 180agctgatcat ggttggccta tatcagattg cactgcagaa ggactaaagg cctcattatt 240gctatcaaag atttctccag aaattgtggg cgaatcggtg gaagttaaca gactatatga 300tgctgtcaat tgtttgatgt cttggatgaa tgaaaatggt ggcttcgcaa catatgaact 360ccaaaggttt tatgcctggc ttgagcttat caaccctgcc gagacattcg gagatattgt 420gattgattac ccgtatgtgg aatgtacctc agccgcaatt caggccctga catcatttaa 480aaagctctat cctgggcacc gcaggaaaga tgtagataac tgtatcaaca aagctgctag 540ttacattgag agcatccaaa gaaaagatgg ttcatggtat ggctcttggg ctgtgtgctt 600cacctatggc acatggttcg gagtgaaggg gctactagct gcaggtagaa ccttcaagag 660cagtcctgca atcagaaagg catgtgactt tctgatgtca aaagagcttc ctttcggtgg 720ctggggagaa agctatctgt catctcaaga tcaggtttac accaatcttg aagggaagca 780tactcatgct gtcaacactg gctgggccat gctgactcta attgacgcag gacaggctga 840gagagacccg acgcctctgc atcgagcagc gaaggttttg ataaacttac aatcagagga 900tggggaattt cctcagcaag agatcatggg agtcttcaac aagaactgca tgatcagcta 960ctcccagtat cggaacatct tccctatctg ggcgcttggc gagtaccgct gccgggtgct 1020gggcgcggcc aagaagtagt accgtcttcc ttctctttgg ccgggttacg tgctggaaca 1080gtgtgtttct gtaataatgt tgctaggtgc aggtggagat ctggtagccg tatagatttt 1140tttttaccat ttgatgagta gaggaataaa ctggagaggg gtatatatgt cgcttgtagg 1200gcctgtttgg ttggatacct gaacaccgtg cctggagaaa tggactgcct ggattgagcc 1260tgagaagatt gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1300 8 345 PRT Triticumaestivum 8 His Glu Asp Thr Ala Phe Ala Val Gln Ala Ile Ala Ala Thr AspLeu 1 5 10 15 Ile Glu Glu Phe Ala Pro Thr Leu Lys Leu Ala His Asp PheIle Lys 20 25 30 Asn Ser Gln Val Val Asp Asp Cys Pro Gly Asp Leu Ser TyrTrp Tyr 35 40 45 Arg His Ile Ser Lys Gly Ala Trp Pro Phe Ser Thr Ala AspHis Gly 50 55 60 Trp Pro Ile Ser Asp Cys Thr Ala Glu Gly Leu Lys Ala SerLeu Leu 65 70 75 80 Leu Ser Lys Ile Ser Pro Glu Ile Val Gly Glu Ser ValGlu Val Asn 85 90 95 Arg Leu Tyr Asp Ala Val Asn Cys Leu Met Ser Trp MetAsn Glu Asn 100 105 110 Gly Gly Phe Ala Thr Tyr Glu Leu Gln Arg Phe TyrAla Trp Leu Glu 115 120 125 Leu Ile Asn Pro Ala Glu Thr Phe Gly Asp IleVal Ile Asp Tyr Pro 130 135 140 Tyr Val Glu Cys Thr Ser Ala Ala Ile GlnAla Leu Thr Ser Phe Lys 145 150 155 160 Lys Leu Tyr Pro Gly His Arg ArgLys Asp Val Asp Asn Cys Ile Asn 165 170 175 Lys Ala Ala Ser Tyr Ile GluSer Ile Gln Arg Lys Asp Gly Ser Trp 180 185 190 Tyr Gly Ser Trp Ala ValCys Phe Thr Tyr Gly Thr Trp Phe Gly Val 195 200 205 Lys Gly Leu Leu AlaAla Gly Arg Thr Phe Lys Ser Ser Pro Ala Ile 210 215 220 Arg Lys Ala CysAsp Phe Leu Met Ser Lys Glu Leu Pro Phe Gly Gly 225 230 235 240 Trp GlyGlu Ser Tyr Leu Ser Ser Gln Asp Gln Val Tyr Thr Asn Leu 245 250 255 GluGly Lys His Thr His Ala Val Asn Thr Gly Trp Ala Met Leu Thr 260 265 270Leu Ile Asp Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Leu His Arg 275 280285 Ala Ala Lys Val Leu Ile Asn Leu Gln Ser Glu Asp Gly Glu Phe Pro 290295 300 Gln Gln Glu Ile Met Gly Val Phe Asn Lys Asn Cys Met Ile Ser Tyr305 310 315 320 Ser Gln Tyr Arg Asn Ile Phe Pro Ile Trp Ala Leu Gly GluTyr Arg 325 330 335 Cys Arg Val Leu Gly Ala Ala Lys Lys 340 345 9 1457DNA Zea mays 9 caaagatgga agaaacacaa agcttgctta tcccttagag aagttccattctgatgttgc 60 tggtaggagc tttcacaatg ggaggtttat acagaggatg cgtcagaaagctgcgtcttt 120 gcccaatgtt caattagaac aaggaactgt tacatcactt ctcgaagaaaatggtactgt 180 taaaggtgtt caatacaaaa ccaagtcagg tgaagaacta aaagcttatgcgcccttgac 240 gattgtatgt gatggctgct tttcaaatct acggcgtgcc ctttgctctccaaaagttga 300 tgttccatca tgttttgttg ggctggtatt ggagaattgc caacttccacatccaaacca 360 tggccatgtt atcttggcca atccttcgcc aatactattt tacccaattagcagcacaga 420 ggtgcgctgt ttggttgatg tcccaggtca gaaggtgcct tccatagctagcggtgaaat 480 ggcaaattat ctcaaaaccg tcgttgcacc ccagattcct ccagaaatctatgactcttt 540 catagcggcc attgataagg gaagcataag aacaatgcca aacaggagcatgccagcggc 600 tccacttcct acccctggcg cacttctgat gggggatgcc ttcaatatgagacacccttt 660 aactggtgga ggaatgactg ttgcattatc cgacatcgtt gtcctacgtaatcttctcaa 720 gcctctccgc aatctccacg acgcatcttc cctgtgcaag tacctcgaatcgttctatac 780 gctgcggaag ccggttgcct ccaccataaa cacgttggcc ggtgctctgtacaaggtctt 840 cagcgcctcg cctgatcaag ctaggaacga gatgcgccag gcctgttttgattacttgag 900 cctcggaggc gtcttctcga atgggcctat tgccttactc tcgggtcttaatcctcggcc 960 actgagttta gttgcacact tcttcgctgt cgctatctac ggtgttggtcgcttgatgct 1020 ccctcttcct tcgcctaaac ggatgtggat tggagccagg ctgatttctggtgcatgcgg 1080 catcatcctc ccgatcatca aagctgaagg cgtgagacag atgttcttccctgccactgt 1140 gcccgcatat taccgggctg cgcctacggg agaataagta aagcgagaaccgattccggg 1200 ctgctgcagt gctgctgatc ccaccatgat ctgatggcaa ctgatgtgtcatggatggca 1260 tttttttcct gtgttagtgg ttgttaggtg gtttgttgtg ctgctgtcattggaatgagg 1320 aacctgtata gtgtgcccct gggtactggt caaagttggg aaatatgttgggtcctaccg 1380 tagccatgtc gattactgcc aagctgtata tgattctgtc gaactagtgaaacctctccc 1440 catctttaac gcgtatc 1457 10 386 PRT Zea mays 10 Asp GlyArg Asn Thr Lys Leu Ala Tyr Pro Leu Glu Lys Phe His Ser 1 5 10 15 AspVal Ala Gly Arg Ser Phe His Asn Gly Arg Phe Ile Gln Arg Met 20 25 30 ArgGln Lys Ala Ala Ser Leu Pro Asn Val Gln Leu Glu Gln Gly Thr 35 40 45 ValThr Ser Leu Leu Glu Glu Asn Gly Thr Val Lys Gly Val Gln Tyr 50 55 60 LysThr Lys Ser Gly Glu Glu Leu Lys Ala Tyr Ala Pro Leu Thr Ile 65 70 75 80Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg Ala Leu Cys Ser Pro 85 90 95Lys Val Asp Val Pro Ser Cys Phe Val Gly Leu Val Leu Glu Asn Cys 100 105110 Gln Leu Pro His Pro Asn His Gly His Val Ile Leu Ala Asn Pro Ser 115120 125 Pro Ile Leu Phe Tyr Pro Ile Ser Ser Thr Glu Val Arg Cys Leu Val130 135 140 Asp Val Pro Gly Gln Lys Val Pro Ser Ile Ala Ser Gly Glu MetAla 145 150 155 160 Asn Tyr Leu Lys Thr Val Val Ala Pro Gln Ile Pro ProGlu Ile Tyr 165 170 175 Asp Ser Phe Ala Ala Ile Asp Lys Gly Ser Ile ArgThr Met Pro Asn 180 185 190 Arg Ser Met Pro Ala Ala Pro Leu Pro Thr ProGly Ala Leu Leu Met 195 200 205 Gly Asp Ala Phe Asn Met Arg His Pro LeuThr Gly Gly Gly Met Thr 210 215 220 Val Ala Leu Ser Asp Ile Val Val LeuArg Asn Leu Leu Lys Pro Leu 225 230 235 240 Arg Asn Leu His Asp Ala SerSer Leu Cys Lys Tyr Leu Glu Ser Phe 245 250 255 Tyr Thr Leu Arg Lys ProVal Ala Ser Thr Ile Asn Thr Leu Ala Gly 260 265 270 Ala Leu Tyr Lys ValPhe Ser Ala Ser Pro Asp Gln Ala Arg Asn Glu 275 280 285 Met Arg Gln AlaCys Phe Asp Tyr Leu Ser Leu Gly Gly Val Phe Ser 290 295 300 Asn Gly ProIle Ala Leu Leu Ser Gly Leu Asn Pro Arg Pro Leu Ser 305 310 315 320 LeuVal Ala His Phe Phe Ala Val Ala Ile Tyr Gly Val Gly Arg Leu 325 330 335Met Leu Pro Leu Pro Ser Pro Lys Arg Met Trp Ile Gly Ala Arg Leu 340 345350 Ile Ser Gly Ala Cys Gly Ile Ile Leu Pro Ile Ile Lys Ala Glu Gly 355360 365 Val Arg Gln Met Phe Phe Pro Ala Thr Val Pro Ala Tyr Tyr Arg Ala370 375 380 Ala Pro 385 11 1289 DNA Oryza sativa 11 aagaagatggtacagttaag ggtgttaaat acaagaccaa gtcaggtgaa gaattaaaag 60 catatgcacctctgacaatt gtatgcgatg gctgtttctc aaaccgcacg agcttacatt 120 gctctccaaaggttgatgta ccatcttgtt ttgttgggct ggtcctggag aattgtcaac 180 ttcctcatgcaaaccatggc catgttgtcc tggccaatcc ttcacctatc ctattttacc 240 caataagcagcactgaagtt cgctgtttgg ttgatgtccc tggtcagaag gtgccttcca 300 tagcaaacggtgaaatggca aaatatctca aaacagtggt tgcacctcag attcctccag 360 aaatctatgattcattcata gcagccattg ataagggaag cataagaaca atgccaaaca 420 ggagcatgccggctgctcca catccaaccc ctggtgcact tttgatgggt gatgcattca 480 acatgcggcatcctttgact ggtggcggaa tgactgttgc attatctgac attgttgtgc 540 tacgtaatcttctcaagcct ctccgcaatc tgcatgatgc atctgctctt tgcaaatacc 600 ttgaatcattctatacactg cggaagccgg ttgcttctac cataaacaca ttagctggtg 660 ctctatacaaggttttcagt gcctcacctg atcaggctag gaatgagatg cgccaagcct 720 gctttgattacttgagcctt ggaggtgtct tttcaaatgg gcctactgct cttctgtctg 780 gtctgaatcctcgaccattg agtttagtgg cacatttctt tgctgtcgct atctatggtg 840 tcggtcgcctaatgcttccc ctcccttcac ctaaacgcat gtggatcggc gtaagactga 900 tttccagtgcatgtggtata attttcccca tcatcaaagc tgaaggtgtg aggcatatgt 960 tcttccccgccactgtccct gcctattatc gtgctcctcg tccaatggag taagggggga 1020 aaatgaaaggagaagcgaag agaaaatccc tgccactgtc ctgatcggcg gatgtttttc 1080 gtggatggcaattttcctgt gtaattggta gtagtcgtca ggccgtgagg ttgtgtgtgc 1140 tgttgttctaatggaacgag gggacctgta tacaccgtca cattccctgt acacttgcca 1200 ctttcgttgttccgtcaggg atgcatgtcg actgctaatc cttaagctgt atatccccca 1260 tgaattcatcatgattgcgt ctttgctct 1289 12 330 PRT Oryza sativa 12 Gly Thr Val Lys GlyVal Lys Tyr Lys Thr Lys Ser Gly Glu Glu Leu 1 5 10 15 Lys Ala Tyr AlaPro Leu Thr Ile Val Cys Asp Gly Cys Phe Ser Asn 20 25 30 Arg Thr Ser LeuHis Cys Ser Pro Lys Val Asp Val Pro Ser Cys Phe 35 40 45 Val Gly Leu ValLeu Glu Asn Cys Gln Leu Pro His Ala Asn His Gly 50 55 60 His Val Val LeuAla Asn Pro Ser Pro Ile Leu Phe Tyr Pro Ile Ser 65 70 75 80 Ser Thr GluVal Arg Cys Leu Val Asp Val Pro Gly Gln Lys Val Pro 85 90 95 Ser Ile AlaAsn Gly Glu Met Ala Lys Tyr Leu Lys Thr Val Val Ala 100 105 110 Pro GlnIle Pro Pro Glu Ile Tyr Asp Ser Phe Ile Ala Ala Ile Asp 115 120 125 LysGly Ser Ile Arg Thr Met Pro Asn Arg Ser Met Pro Ala Ala Pro 130 135 140His Pro Thr Pro Gly Ala Leu Leu Met Gly Asp Ala Phe Asn Met Arg 145 150155 160 His Pro Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ser Asp Ile Val165 170 175 Val Leu Arg Asn Leu Leu Lys Pro Leu Arg Asn Leu His Asp AlaSer 180 185 190 Ala Leu Cys Lys Tyr Leu Glu Ser Phe Tyr Thr Leu Arg LysPro Val 195 200 205 Ala Ser Thr Ile Asn Thr Leu Ala Gly Ala Leu Tyr LysVal Phe Ser 210 215 220 Ala Ser Pro Asp Gln Ala Arg Asn Glu Met Arg GlnAla Cys Phe Asp 225 230 235 240 Tyr Leu Ser Leu Gly Gly Val Phe Ser AsnGly Pro Thr Ala Leu Leu 245 250 255 Ser Gly Leu Asn Pro Arg Pro Leu SerLeu Val Ala His Phe Phe Ala 260 265 270 Val Ala Ile Tyr Gly Val Gly ArgLeu Met Leu Pro Leu Pro Ser Pro 275 280 285 Lys Arg Met Trp Ile Gly ValArg Leu Ile Ser Ser Ala Cys Gly Ile 290 295 300 Ile Phe Pro Ile Ile LysAla Glu Gly Val Arg His Met Phe Phe Pro 305 310 315 320 Ala Thr Val ProAla Tyr Tyr Arg Ala Pro 325 330 13 1883 DNA Glycine max 13 gcacgaggaaactagagcca gaaagagaaa caaagagagc gagagcgaga gcgaaaacac 60 ctcaacgtcgtcgtcgtagc cgagaggttc ctcgcaatgg tggaccccta cgtgctcgga 120 tggatcatatgcgccgtgct cagcctcgtc gcgcttcgca atttcgcttt cgcgcggaag 180 aaccgttgccattcgtctga gaccgatgcc actcgccgcg cggaaaatgt caccaccgcc 240 gccggagaatgcagatcctc gagtcgcgac ggcgacgttg acgtcattat tgtcggagct 300 ggtgtcgccggctccgctct cgctcacact ctcggcaagg atgggcgtcg ggtacttgtc 360 attgaaagagatttgagtga acaagaccga attgttgggg agttgctaca acctggaggc 420 tatctcaaattaattgagct gggacttgaa gattgtgtgg agaaaattga tgctcaacta 480 gtgtttggttatgctctttt caaggatggg aagcacacaa gactctctta tcccttggaa 540 aagtttcactcagatgttgc tggcagaagc tttcacaatg ggcgttttat tcagaggatg 600 agagagaaggctgcctccct ttccaatgta cgactggagc aaggaacagt cacttcccta 660 cttgaagagaagggggttat taaaggtgtg cactacaaaa cgaaggatag tcaagaatta 720 tcagcttgtgcaccccttac cgttgtttgt gatggctgtt tctcaaactt gcgccgatct 780 ctttgtaatcctaaggtaga tgttccctct catttcgttg gcttaatttt ggagagttgt 840 gaacttccttatgctaatca tggccatgtc atactgggag atccttcgcc agttctgttc 900 tatcggataagtagttcaga aattcgttgt ctggttgatg ttcctggtca gaaggttcca 960 tctatttcgaatggtgaaat gacaaattat ttgaagacag tggtagctcc acagattcca 1020 cctgagcttcatgactcatt cgtagctgca gtggacaaag gcaacatcag gacaatgcca 1080 aacagaagcatgccagcagc tccttatcct acgcccggag ccctgttgat gggagatgca 1140 ttcaacatgcgccatcctct aaccgggggt ggaatgactg tggcattatc tgacatagta 1200 gtgctgcgaaatcttctgag acctttgcgt gacctgaatg atgcacctgg cctttgcaaa 1260 tacctagaatccttttatac cttacgcaag cctgtggcat ccactataaa tacgttggca 1320 ggagcactttacaaggtttt ttgcgcatca cctgatccag caaggaagga aatgcgccaa 1380 gcttgcttcgattatcttag tcttggaggt ctattctcgg aagggccagt ctctttgctt 1440 tcaggattaaaccctcggcc cttgagcctg gttctccatt tctttgctgt tgcaatatat 1500 ggtgttggccgtttactgct accatttcct tcacctaaac ggatgtggat tggagtccga 1560 ttaatttctagtgcatctgg aatcatcttg ccaataatta aggcagaagg agtccgtcag 1620 atgttcttccctgcaactgt tccagcttac tatagaaatc ccccggccca ataaatgtga 1680 gttccgtgaacccatcatga gtcattcaag atgagccacc agtgttttcc attcagaaaa 1740 ttaacgggttcaattgagat gtttgcaaac aatctggctt tagtgtcatg taaagtcgat 1800 tttaaattaaatgtttgatt tgttaatctt cttaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860 aaaaaaaaaaaaaaaaaaaa aaa 1883 14 523 PRT Glycine max 14 Met Val Asp Pro Tyr ValLeu Gly Trp Ile Ile Cys Ala Val Leu Ser 1 5 10 15 Leu Val Ala Leu ArgAsn Phe Ala Phe Ala Arg Lys Asn Arg Cys His 20 25 30 Ser Ser Glu Thr AspAla Thr Arg Arg Ala Glu Asn Val Thr Thr Ala 35 40 45 Ala Gly Glu Cys ArgSer Ser Ser Arg Asp Gly Asp Val Asp Val Ile 50 55 60 Ile Val Gly Ala GlyVal Ala Gly Ser Ala Leu Ala His Thr Leu Gly 65 70 75 80 Lys Asp Gly ArgArg Val Leu Val Ile Glu Arg Asp Leu Ser Glu Gln 85 90 95 Asp Arg Ile ValGly Glu Leu Leu Gln Pro Gly Gly Tyr Leu Lys Leu 100 105 110 Ile Glu LeuGly Leu Glu Asp Cys Val Glu Lys Ile Asp Ala Gln Leu 115 120 125 Val PheGly Tyr Ala Leu Phe Lys Asp Gly Lys His Thr Arg Leu Ser 130 135 140 TyrPro Leu Glu Lys Phe His Ser Asp Val Ala Gly Arg Ser Phe His 145 150 155160 Asn Gly Arg Phe Ile Gln Arg Met Arg Glu Lys Ala Ala Ser Leu Ser 165170 175 Asn Val Arg Leu Glu Gln Gly Thr Val Thr Ser Leu Leu Glu Glu Lys180 185 190 Gly Val Ile Lys Gly Val His Tyr Lys Thr Lys Asp Ser Gln GluLeu 195 200 205 Ser Ala Cys Ala Pro Leu Thr Val Val Cys Asp Gly Cys PheSer Asn 210 215 220 Leu Arg Arg Ser Leu Cys Asn Pro Lys Val Asp Val ProSer His Phe 225 230 235 240 Val Gly Leu Ile Leu Glu Ser Cys Glu Leu ProTyr Ala Asn His Gly 245 250 255 His Val Ile Leu Gly Asp Pro Ser Pro ValLeu Phe Tyr Arg Ile Ser 260 265 270 Ser Ser Glu Ile Arg Cys Leu Val AspVal Pro Gly Gln Lys Val Pro 275 280 285 Ser Ile Ser Asn Gly Glu Met ThrAsn Tyr Leu Lys Thr Val Val Ala 290 295 300 Pro Gln Ile Pro Pro Glu LeuHis Asp Ser Phe Val Ala Ala Val Asp 305 310 315 320 Lys Gly Asn Ile ArgThr Met Pro Asn Arg Ser Met Pro Ala Ala Pro 325 330 335 Tyr Pro Thr ProGly Ala Leu Leu Met Gly Asp Ala Phe Asn Met Arg 340 345 350 His Pro LeuThr Gly Gly Gly Met Thr Val Ala Leu Ser Asp Ile Val 355 360 365 Val LeuArg Asn Leu Leu Arg Pro Leu Arg Asp Leu Asn Asp Ala Pro 370 375 380 GlyLeu Cys Lys Tyr Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val 385 390 395400 Ala Ser Thr Ile Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Cys 405410 415 Ala Ser Pro Asp Pro Ala Arg Lys Glu Met Arg Gln Ala Cys Phe Asp420 425 430 Tyr Leu Ser Leu Gly Gly Leu Phe Ser Glu Gly Pro Val Ser LeuLeu 435 440 445 Ser Gly Leu Asn Pro Arg Pro Leu Ser Leu Val Leu His PhePhe Ala 450 455 460 Val Ala Ile Tyr Gly Val Gly Arg Leu Leu Leu Pro PhePro Ser Pro 465 470 475 480 Lys Arg Met Trp Ile Gly Val Arg Leu Ile SerSer Ala Ser Gly Ile 485 490 495 Ile Leu Pro Ile Ile Lys Ala Glu Gly ValArg Gln Met Phe Phe Pro 500 505 510 Ala Thr Val Pro Ala Tyr Tyr Arg AsnPro Pro 515 520 15 1948 DNA Triticum aestivum 15 gcacgagctc tcgtctcgtctcgtctctcg tcccaatccc atcgcccggc actctccccc 60 gctcgtctcc tccccgcacgcatcaccctc tctcccctcg cccggtcgaa gggatccccg 120 cgccggagga cggctgcgcggtcgctgacg gcgcagggag cgcggccgtg gacggcccga 180 cggacgtcat catcgtcggagccggggtcg ccggatctgc cctcgcctac acgctcggaa 240 aggatggtcg acgggtgcatgtcatagaga gagacctgac agagcctgat agaattgtgg 300 gtgaattgtt acaacctggaggctacctga aattgatgga attgggtctg caggactgcg 360 ttgatgaaat tgatgcacagcgtgtccttg gttatgcatt attcaaagat gggaagaaca 420 caaaactttc ttaccccttggagaagttcc attcagatgt ggctggcagg agctttcaca 480 atggacggtt catacagaggatgcgagaaa aggctgcatc tttgcccaat gtccaactgg 540 agcaaggaac agttacatctttgcttgaag aaaatggtac agttaagggt gtgcaataca 600 agatcaagtc aggtgaagaactaaaagctt atgcaccatt gacaattgta tgtgatggct 660 gcttttcaaa cttaagacgtgccctttgct ctccaaaggt tgaggtgccg tcttgctttg 720 ttggcctggt cttggagaattgtgaacttc ctcatgcgaa ccatggccat gttatcttgg 780 ccaatccttc tcccatcctattttacccga taagcagcac cgaggttcgc tgtttggtag 840 atgtccctgg tcagaaggtgccttccatag caagtggtga aatgacaaat tatctcaaga 900 ccgtggttgc acctcagattcctccacaaa tctgtgattc ttttatagca gcaattgata 960 agggaagcat aagaacaatgccaaatagga gcatgccagc tgcaccacat ccaacacctg 1020 gtgcactttt gatgggagatgctttcaata tgcgacaccc tttaacaggt ggaggaatga 1080 ctgttgcatt atcagatatagtcgtcctgc gtaatcttat caagcttctt cgcaatctgc 1140 atgatgcctc tgccctctgcaaatacctcg agtcattcta tactctgcgg aagccggttg 1200 cttctacaat aaacacattggctggtgctc tatacaaagt cttcagttcc tcgcctgaca 1260 aggctaggga tgagatgcgccaagcttgct ttgattactt gagccttgga ggtgtctgtt 1320 caaatgggcc cattgctctactctccggtc ttaatcctcg gccattgagt ttggttgcac 1380 acttctttgc tgttgctatctttggtgttg gacgactgat gctccccctt ccttcaccta 1440 aacgaatgtg gactggagcgagattgattt caggtgcatg tggtatcatc ttcccaatca 1500 tcaaagctga aggtgtgaggcaaatgttct tccctgctac cgtccccgcg tattaccggg 1560 ctcctcccga agcggagttctgaatgacga aggtgcagct aatctctctt gcacgatgac 1620 atttttccct gtgtcggtagtcgtctacag tgttagccgg tcactggaat gtgctgtgtt 1680 ggtagtctga atggatcgaggaacgtgtat agtatctccg ctgggtgctg atcctgtttt 1740 tgaaatgttt tgaattgctgcgtcggtgcc catgttgatt cgtcctagtg aaattgtaca 1800 tctttgttgt actacgccctccgttccgaa ttacttgtcg cacttatgga tgtatcaaga 1860 tgtattttag ttctagatacatccatttta acgacgagta atttggaaaa aaaaaaaaaa 1920 aaaaaaaaaa aaaaaaaaaaaaaaaaaa 1948 16 467 PRT Triticum aestivum 16 Val Asp Gly Pro Thr AspVal Ile Ile Val Gly Ala Gly Val Ala Gly 1 5 10 15 Ser Ala Leu Ala TyrThr Leu Gly Lys Asp Gly Arg Arg Val His Val 20 25 30 Ile Glu Arg Asp LeuThr Glu Pro Asp Arg Ile Val Gly Glu Leu Leu 35 40 45 Gln Pro Gly Gly TyrLeu Lys Leu Met Glu Leu Gly Leu Gln Asp Cys 50 55 60 Val Asp Glu Ile AspAla Gln Arg Val Leu Gly Tyr Ala Leu Phe Lys 65 70 75 80 Asp Gly Lys AsnThr Lys Leu Ser Tyr Pro Leu Glu Lys Phe His Ser 85 90 95 Asp Val Ala GlyArg Ser Phe His Asn Gly Arg Phe Ile Gln Arg Met 100 105 110 Arg Glu LysAla Ala Ser Leu Pro Asn Val Gln Leu Glu Gln Gly Thr 115 120 125 Val ThrSer Leu Leu Glu Glu Asn Gly Thr Val Lys Gly Val Gln Tyr 130 135 140 LysIle Lys Ser Gly Glu Glu Leu Lys Ala Tyr Ala Pro Leu Thr Ile 145 150 155160 Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg Ala Leu Cys Ser Pro 165170 175 Lys Val Glu Val Pro Ser Cys Phe Val Gly Leu Val Leu Glu Asn Cys180 185 190 Glu Leu Pro His Ala Asn His Gly His Val Ile Leu Ala Asn ProSer 195 200 205 Pro Ile Leu Phe Tyr Pro Ile Ser Ser Thr Glu Val Arg CysLeu Val 210 215 220 Asp Val Pro Gly Gln Lys Val Pro Ser Ile Ala Ser GlyGlu Met Thr 225 230 235 240 Asn Tyr Leu Lys Thr Val Val Ala Pro Gln IlePro Pro Gln Ile Cys 245 250 255 Asp Ser Phe Ile Ala Ala Ile Asp Lys GlySer Ile Arg Thr Met Pro 260 265 270 Asn Arg Ser Met Pro Ala Ala Pro HisPro Thr Pro Gly Ala Leu Leu 275 280 285 Met Gly Asp Ala Phe Asn Met ArgHis Pro Leu Thr Gly Gly Gly Met 290 295 300 Thr Val Ala Leu Ser Asp IleVal Val Leu Arg Asn Leu Ile Lys Leu 305 310 315 320 Leu Arg Asn Leu HisAsp Ala Ser Ala Leu Cys Lys Tyr Leu Glu Ser 325 330 335 Phe Tyr Thr LeuArg Lys Pro Val Ala Ser Thr Ile Asn Thr Leu Ala 340 345 350 Gly Ala LeuTyr Lys Val Phe Ser Ser Ser Pro Asp Lys Ala Arg Asp 355 360 365 Glu MetArg Gln Ala Cys Phe Asp Tyr Leu Ser Leu Gly Gly Val Cys 370 375 380 SerAsn Gly Pro Ile Ala Leu Leu Ser Gly Leu Asn Pro Arg Pro Leu 385 390 395400 Ser Leu Val Ala His Phe Phe Ala Val Ala Ile Phe Gly Val Gly Arg 405410 415 Leu Met Leu Pro Leu Pro Ser Pro Lys Arg Met Trp Thr Gly Ala Arg420 425 430 Leu Ile Ser Gly Ala Cys Gly Ile Ile Phe Pro Ile Ile Lys AlaGlu 435 440 445 Gly Val Arg Gln Met Phe Phe Pro Ala Thr Val Pro Ala TyrTyr Arg 450 455 460 Ala Pro Pro 465

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having squalene monooxygenaseactivity, wherein the amino acid sequence of the polypeptide and theamino acid sequence of SEQ ID NO:14 have at least 90% identity based onthe Clustal method of alignment, or (b) the complement of the nucleotidesequence, wherein the complement and the nucleotide sequence contain thesame number of nucleotides and are 100% complementary.
 2. Thepolynucleotide of claim 1, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:14 have at least95% identity based on the Clustal alignment method.
 3. Thepolynucleotide of claim 1, wherein the polypeptide comprises the aminoacid sequence of SEQ ID NO:14.
 4. The polynucleotide of claim 1, whereinthe nucleotide sequence comprise the nucleotide sequence of SEQ IDNO:13.
 5. A cell comprising the polynucleotide of claim
 1. 6. The cellof claim 5, wherein the cell is selected from the group consisting of ayeast cell, a bacterial cell and a plant cell.
 7. A transgenic plantcomprising the polynucleotide of claim
 1. 8. A method for transforming acell comprising introducing into a cell the polynucleotide of claim 1.9. A method for producing a transgenic plant comprising (a) transforminga plant cell with the polynucleotide of claim 1, and (b) regenerating aplant from the transformed plant cell.
 10. A chimeric gene comprisingthe polynucleotide of claim 1 operably linked to at least one regulatorysequence.
 11. A vector comprising the polynucleotide of claim
 1. 12. Aseed comprising the chimeric gene of claim
 10. 13. A method forisolating a polypeptide encoded by the polynucleotide of claim 1comprising isolating the polypeptide from a cell containing a chimericgene comprising the polynucleotide operably linked to a regulatorysequence.